Project Summary/Abstract Because mitochondria have vital roles in cell metabolism and other functions, it is critical to maintain the quality of the mitochondrial population within cells. Mitophagy, the degradation of mitochondria via autophagy, is thought to be the major pathway for culling dysfunctional or damaged mitochondria. The best- studied mitophagy pathway involves Pink1 and Parkin, but questions remain about the physiological importance of this pathway. Mouse knockouts of Pink1 and Parkin show only subtle defects in basal mitochondrial function, inconsistent with a widespread defect in mitophagy. This observation suggests that there are alternative or redundant pathways for mitophagy. Our preliminary data indicate that Fis1, a protein previously thought to be involved in mitochondrial fission, is a major player in mitophagy, including mitophagy that is Pink1/Parkin-independent. Moreover, mice lacking Fis1 have severe physiological defects. Therefore, the study of Fis1 mutant mice may reveal the physiological functions of mitophagy. The long-term goals of this research project are to understand the molecular mechanism of Fis1-dependent mitophagy and the role of mitophagy in mouse development. In Aim 1, we develop a functional assay to dissect the molecular mechanism underlying uniparental inheritance of mitochondria in mammals. Using this system, we will identify molecules involved in elimination of paternal mitochondria in the early mouse embryo. In Aim 2, we develop a cellular system in which increased oxidative phosphorylation stimulates mitophagy. We will use this cellular system to understand the molecular mechanism of mitophagy, including the role of Fis1, the role of ubiquitination, and the recruitment of the autophagy adaptor p62. In Aim 3, we utilize a Fis1 knockout mouse model to understand the physiological functions of mitophagy. We will study the role of Fis1 in male germ cell maturation, where loss of Fis1 results in increased mitochondrial and peroxisomal density. Taken together, these Aims will lead to a deep understanding of the in vivo roles of Fis1 and the role of mitophagy in mammalian physiology.